Method of cryopreserving selected sperm cells

ABSTRACT

The present invention provides a method of cryopreserving sperm that have been selected for a specific characteristic. In a preferred embodiment, the method is employed to freeze sex-selected sperm. Although the cryopreservation method of the invention can be used to freeze sperm selected by any number of selection methods, selection using flow cytometry is preferred. The present invention also provides a frozen sperm sample that has been selected for a particular characteristic, such as sex-type. In preferred embodiments, the frozen sperm sample includes mammalian sperm, such as, for example, human, bovine, equine, porcine, ovine, elk, or bison sperm. The frozen selected sperm sample can be used in a variety of applications. In particular, the sample can be thawed and used for fertilization. Accordingly, the invention also includes a method of using the frozen selected sperm sample for artificial insemination or in vitro fertilization.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/266,562, filed Oct. 7, 2002, which is a continuation of U.S. patentapplication Ser. No. 09/577,246, filed May 24, 2000, which is adivisional of U.S. patent application Ser. No. 09/478,299 filed Jan. 5,2000 which claims the benefit of U.S. Provisional Application No.60/167,423, filed Nov. 24, 1999, each hereby incorporated by referenceherein.

FIELD OF THE INVENTION

The invention relates to a method for freezing sperm selected for aparticular characteristic, as well as to a frozen selected sperm sampleand methods of using such a sample. The invention is particularly usefulfor preserving sex-selected sperm.

BACKGROUND OF THE INVENTION

Over half a century ago, artificial insemination was introduced in theUnited States as a commercial breeding tool for a variety of mammalianspecies. Although artificial insemination was initially limited toregions relatively close to the site of sperm collection, advances inthe cryopreservation and storage of sperm have facilitated widespreaddistribution and commercialization of sperm intended for artificialinsemination or in vitro fertilization.

Further improvements in mammalian sperm collection, selection,cryopreservation, storage, and handling techniques have enhanced theability of breeders to produce animals having desired traits. Forexample, advances in selection of mammalian sperm based on slightdifferences in physical characteristics has made it possible to separatesperm based on sex-type, that is, to select for cells containing eitherthe X or Y chromosome. This technique allows the breeder to manipulatethe relative percentage of X- or Y-type sperm in a sample and therebydetermine offspring sex. The ability to select sperm based on sex-typeor any other desirable characteristic provides an important tool foraccelerating genetic progress, increasing production efficiency, andachieving greater flexibility in livestock management. Full exploitationof this tool, however, depends on the ability to freeze and storeselected sperm.

A variety of methods are available for selecting cells; however, theselection and subsequent processing of sperm presents unique challengesbecause sperm are incapable of DNA repair and because of spermmorphology. Each sperm has an acrosome overlying the head and a tail,which are important for fertility and which are relatively susceptibleto physical injury. In addition, sperm fertility decreases withincreasing time between collection and use. As most of the availableselection methods involve physical stresses and take time, selectedsperm are typically somewhat compromised compared to non-selected cells.Fertility may be further reduced if the selection technique involvessignificant dilution. It has been suggested that this “dilution effect”may be due to the loss of protective components in seminal plasma.

Flow cytometry is a particularly efficient selection method that hasbeen employed for sorting sperm by sex-type. However, sorted sperm aresubject to stresses beyond those normally encountered in standardartificial insemination or in vitro fertilization protocols. Inparticular, flow cytometry is time consuming, and, because of thephysical constraints of flow cytometers, sperm must be diluted forsorting to levels that are not optimal for storage. (usually to on theorder of 10⁵-10⁶/ml). Furthermore, sorted sperm intended for artificialinsemination must be concentrated so that conventional packaging anddelivery equipment can be used. The need for a concentration step thusexposes already somewhat compromised sperm to additional physicalstresses.

The freezing of sperm also invariably reduces fertility, motility,and/or viability, and, although techniques for freezing unselected spermare well known, no technique for cryopreservation of selected sperm hasbeen described.

SUMMARY OF THE INVENTION

The present invention provides a method of cryopreserving sperm thathave been selected for a specific characteristic. The method isparticularly useful for cryopreserving sperm selected by a method thatresults in dilution of the sperm, since the method provides for theisolation of sperm from a selected sperm sample, followed by addition ofa final extender to the isolated sperm to produce a suspension having adesired concentration of sperm. In a preferred embodiment, the method isemployed to freeze sex-selected sperm. Although the cryopreservationmethod of the invention can be used to freeze sperm selected by anynumber of selection methods, selection using flow cytometry ispreferred.

The present invention also provides a frozen sperm sample that has beenselected for a particular characteristic, such as sex-type. In preferredembodiments, the frozen sperm sample includes mammalian sperm, such as,for example, human, bovine, equine, porcine, ovine, elk, or bison sperm.Also within the scope of the invention is a container including a frozensperm sample according to the invention.

The frozen selected sperm sample can be used in a variety ofapplications. In particular, the sample can be thawed and used forfertilization. Accordingly, the invention also includes a method ofusing the frozen selected sperm sample for artificial insemination or invitro fertilization.

DETAILED DESCRIPTION OF THE INVENTION

The present invention allows cryopreservation of sperm that have beenselected for a particular characteristic, facilitating storage and/orshipment of selected sperm samples to sites distant from the collectionsite. Thawing yields viable sperm that can be used in procedures such asartificial insemination (“AI”) and in vitro fertilization (“IVF”). Thisresult was surprising because of the well-documented fragility of sperm.Prior researchers had demonstrated that the stresses associated withvarious selection methods or with cryopreservation resulted insignificant losses in fertility and/or viability. The present inventorshave demonstrated, for the first time, that pregnancies can be achievedwith sperm that have been selected and then frozen.

The invention represents an important advance in livestock management,where selection of sperm for use in such procedures can be used toincrease the production of offspring having desirable traits. Forexample, selection to obtain sperm carrying either the X or the Ychromosome allows control over offspring sex, which is advantageous forproducers of animals such as dairy or beef cattle. Sex selection alsofinds application in breeding valuable (e.g., show or race horses) orendangered animals. The ability to freeze selected sperm, which theinvention provides, will enable widespread use of such selection methodsto, e.g., increase livestock production efficiency as well as quality.

DEFINITIONS

The term “acrosome” or “acrosomal cap” refers to the cap that covers theanterior half of the head of sperm and that contains enzymes necessaryfor ovum penetration.

The term “sex-type” refers to the type of sex chromosome present in thesperm (i.e., the X or Y chromosome).

The term “capacitation” refers to the specific changes a sperm undergoesto develop the capacity to fertilize ova, such as enzymic changes on thesurface of the acrosome that lead to release of acrosomal enzymes thatfacilitate penetration of the sperm into the ovum.

As used with reference to sperm, the term “cryoprotectant” refers to amolecule that protects sperm during a freeze-thaw cycle, promotingsurvival and retention of fertilizing capacity.

The term “dilution effect” refers to the rapid decline in motilityand/or viability of sperm when highly diluted.

As used herein, the term “selection” refers to a method whereby a sampleis subdivided based on presence or absence of a specific characteristic(unless context dictates otherwise). Thus, a “selected sperm sample” isa sample obtained by subjecting a source sample to selection for thespecific characteristic. A selected sperm sample is therefore enriched,relative to the source sample, in sperm having the specificcharacteristic.

The term “sorting” is used herein to describe a selection method carriedout using a fluorescence-activated cell sorter (FACS).

The term “extender” refers to any medium that tends to preserve spermviability. The term “extension” refers to the dilution of sperm withextender.

The term “initial extender” refers to a medium used to extend spermprior to the isolation step of the method of this invention.

The term “final extender” refers to a medium used to extend sperm priorto the freezing step of the method of this invention.

An “organic substance” in an extender described herein is any organicsubstance that tends to reduce cold shock and preserve fertility ofsperm.

An “energy source” in an extender described herein is any substance orsubstrate that sperm can utilize for cell maintenance and/or motility.

The term “osmolality,” as used herein, is a measure of the osmoticpressure of dissolved solute particles in a an aqueous solution (e.g.,an extender). The solute particles include both ions and non-ionizedmolecules. Osmolality is expressed as the concentration of osmoticallyactive particles (i.e., osmoles) dissolved in 1 kg of water.

Cryopreservation Method

The invention provides a method of cryopreserving selected spermincludes the following steps:

(1) obtaining a selected sperm sample;

(2) cooling the selected sperm sample;

(3) isolating sperm from the selected sperm sample;

(4) adding final extender to the isolated sperm to produce a suspensionof sperm; and

(5) freezing the suspension of sperm.

Obtaining a Selected Sperm Sample

The first step in the cryopreservation method of the inventionencompasses obtaining a previously selected sperm sample, as well assubjecting a source sample to any suitable selection method. Sperm fromany species can be selected and frozen according to the method of theinvention. The method can be carried out with sperm from domesticatedanimals, especially livestock, as well as with sperm from wild animals(e.g., endangered species). Preferably, the selected sperm samplecontains mammalian sperm. Human sperm, bovine, equine, porcine, ovine,elk, and bison sperm are particularly preferred.

Generally, the selected sperm sample contains normal, viable sperm. Tothis end, the ejaculate from which the sperm are obtained typically hasat least about 50%, and preferably at least about 75% morphologicallynormal sperm. In these embodiments, generally at least about 40%, andpreferably at least about 60% of the sperm in the ejaculate exhibitprogressive motility.

A wide variety of methods for selecting cells from a mixed populationsare available, including, for example, selection based on binding ofcells or cell components with antibodies, antibody fragments, or otherbinding partners and differential staining.

The invention is exemplified herein with selection based on sex-type,and sex-selected sperm for use in the invention can be obtained usingany selection strategy that takes advantage of slight differences incharacteristics between X- and Y-type sperm. Exemplary sex-selectionmethods include magnetic techniques (see, e.g., U.S. Pat. No.4,276,139), columnar techniques (see, e.g., U.S. Pat. No. 5,514,537)gravimetric techniques (see, e.g., U.S. Pat. No. 3,894,529, reissue Pat.No. 32350, U.S. Pat. Nos. 4,092,229, 4,067,965, and 4,155,831).Sex-selection based on differences in electrical properties is disclosedin U.S. Pat. No. 4,083,957, and techniques that select based ondifferences in electrical and gravimetric properties are discussed inU.S. Pat. Nos. 4,225,405, 4,698,142, and 4,749,458. U.S. Pat. Nos.4,009,260 and 4,339,434 describe selection based on differences inmotility. Biochemical techniques relying on antibodies are disclosed inU.S. Pat. Nos. 4,511,661, 4,999,283, 3,687,806, 4,191,749, 4,448,767,whereas U.S. Pat. Nos. 5,021,244, 5,346,990, 5,439,362, and 5,660,997describe selection based on differences in membrane proteins.

Flow cytometry is a preferred method for separating cells from mixedpopulations based on differential staining with fluorescent dyes orbinding to fluorescently labeled molecules, such as antibodies ornucleic acids. In fluorescence activated cell sorting (“FACS”), cellsare “sorted” into different populations based on the fluorescenceintensity upon irradiation. FACS can be used for sex-selection of spermbecause the X chromosome contains slightly more DNA than the Ychromosome. When sperm are stained with a fluorescent DNA-binding dye,X-chromosome bearing sperm absorb more dye than Y chromosome bearingsperm and the two populations can therefore can be separated by FACS.This strategy was discussed in U.S. Pat. No. 4,362,246 and significantlyexpanded upon in U.S. Pat. No. 5,135,759 (issued to Johnson). Separationhas been enhanced through-the use of high-speed flow cytometers, such asthe MoFlo® flow cytometer produced by Cytomation, Inc. (Ft. Collins,Colo.) and described in U.S. Pat. Nos. 5,150,313, 5,602,039, 5,602,349,and 5,643,796, as well as in PCT Publication No. WO 96/12171.

The selection method used to obtain the selected sperm sample ispreferably one that preserves sperm viability. Because of the relativefragility of sperm, normal flow cytometry techniques should generally bemodified for sorting sperm. More specifically, the flow cytometryentails staining, dilution, and interrogation of cells with light. Allof these steps represent stresses that can reduce sperm viability. Thesensitivity of sperm to these stresses can vary between species and evenbetween individuals within species. Such sensitivities have either beendocumented or can readily be determined by empirical studies, such asthose described in Examples 1-5.

Modifications that enhance viability are described the patentpublications discussed above. For instance, procedures that provideimproved sheath and collector systems for sorting sperm are disclosed inPCT Publication No. WO 99/33956 (Application No. PCT/US98/27909).Further, Examples 1-7 below describe exemplary procedures for stainingand sorting sperm. Example 3 describes a study of the effects of laserintensity and dye concentration of post-thaw motility of sorted frozensperm. This study indicates that the use of lower laser intensitiesduring sorting can increase post-thaw motility.

The selected sperm sample can contain a variety of components besidessperm and will often contain components added to protect the spermduring the selection process. In the case of FACS, the selected spermsample can contain component(s) of the solutions used for staining andsorting (e.g., the sheath fluid and the catch buffer).

In addition, the selected sperm sample typically contains an extender orextender fraction. For example, “two-step” extenders including an “Afraction” lacking glycerol and a “B fraction” containing glycerol arewell known. The A fraction is added to sperm first, followed by additionof an equal volume of the B fraction. For this step, the B fraction isoften divided into at least two aliquots and added sequentially; e.g.,the second B fraction aliquot is added 15 minutes after the first.

If no extender components are present, an extender or extender fractionis typically added to the selected sperm sample before the sperm areisolated from the sample. If only some extender components are present,additional components can optionally be added so that selected spermsample includes a complete extender or an extender fraction before theisolation step. In exemplary embodiments, bovine sperm are flow-sortedso as to produce a selected sperm sample including the A fraction of anextender (see Examples 2, 3, and 4). If desired, the B fraction can thenbe added to the selected sperm sample before the isolation step (seeExample 5). The pre-isolation step extender (or extender fraction) istermed “the initial extender” to distinguish it from the “finalextender” employed for the extension of isolated sperm before freezing.If the selected sperm sample was selected using FACS, the initialextender can be matched to the sheath fluid employed for sorting.Exemplary matched sheath fluids and extenders are described in detail inExample 4.

An extender suitable for use in the selected sperm sample includes aphysiologically acceptable carrier. The physiologically acceptablecarrier is typically aqueous, and, in preferred embodiments, includesdeionized water. Suitable extenders commonly comprise one or more of thefollowing additional components: a cryoprotectant, a component thatmaintains osmolality and buffers pH, an organic substance that preventscold shock and preserves fertility of sperm, a detergent that actssynergistically with certain organic substances to enhance preservationof sperm, an energy source that can be readily utilized by sperm, anantioxidant, which protects sperm from cold shock, a substance thatfacilitates sperm capacitation, and one or more antibiotics.

Although cryoprotectants useful in the invention are not limited tothose acting by a particular mechanism, most conventionalcryoprotectants act, at least in part, by reducing intracellulardehydration. Specifically, freezing is accompanied by an increase insolute concentration in the medium surrounding sperm. This increase insolute concentration draws water out of the cells, which increasesintracellular electrolyte concentration. Exemplary cryoprotectantsinclude glycerol, dimethyl sulfoxide, ethylene glycol, propylene glycol,and the like. The cryoprotectant suitable for use in a given extendercan vary, depending on the species from which sperm are derived. Forexample, glycerol is suitable for use in cryopreservation of human andbovine sperm, but is generally not used for cryopreservation of porcineor rabbit sperm. Such preferences are well known for many commerciallyvaluable sperm and can readily be determined empirically for other typesof sperm.

The extender useful in the invention optionally includes one or morecomponents that help maintain osmolality and provide buffering capacity.In preferred embodiments of the invention, the osmolality of theextender approximates that of physiological fluids. More preferably, theosmolality of the extender is in the range of about 280 mOsm to about320 mOsm. The pH is also preferably within a physiologically acceptablerange, more preferably in the range of about 6.5 to about 7.5.

Substances helpful in maintaining osmolality and pH within these rangesare well known in the art and can be added to the extender as a solid oralready in solution. A buffer containing a salt, a carbohydrate, or acombination thereof can be employed for this purpose. Specific examplesinclude sodium citrate, Tris[hydroxymethyl]aminomethane, and TES(N-Tris[Hydroxymethyl]methyl-2-aminoethanesulfonic acid), andmonosodium. glutamate buffers; milk; HEPES-buffered medium; and anycombination thereof. The component employed to help maintain osmolalityand provide buffering capacity in a particular application can varydepending on the other components of the extender and, in some cases, onthe species from which the sperm are derived. The selection of such acomponent for use in the present invention is, however, within the levelof skill in the art.

One or more organic substances that protect sperm against cold shock andhelp preserve fertilizing capacity can also be included in the extender.Such substances are well known and are sometimes described as“nonpenetrating cryoprotectants.” One skilled in the art can readilydetermine an organic substance suitable for a particular application ofthe cryopreservation method described herein. For example, organicsubstances containing protective constituents (e.g., lipoproteins,phospholipids, lecithin) that are believed to reduce the impact of coldshock and the dilution effect can be included in the extender. Suitableorganic substances include disaccharides, trisaccharides, and anycombination thereof. Exemplary organic substances include egg yolk, anegg yolk extract, milk, a milk extract, casein, albumin, lecithin,cholesterol, and any combination thereof.

The extender can also include a detergent. Alkyl ionic detergents, suchas sodium dodecyl sulfate (SDS), have been reported to actsynergistically with egg yolk to enhance protection against cold shock.Other detergents useful in the cryopreservation of cells can also beemployed in the extender, and the selection of a particular detergentfor a specific application is within the level of skill in the art inlight of the guidance provided herein. See, e.g., Example 5.

Preferably, the extender includes an energy source that is readilyutilized by sperm. In the absence of an energy source, sperm may oxidizeintracellular phospholipids and other cellular components. Thus, theinclusion of an energy source in the extender protects intracellularreserves and cellular components. As is well known in the art, sugars,particularly monosaccharides, provide a convenient energy source,although any conventional energy source can be employed in the extender.Exemplary monosaccharides useful in the extender include glucose,fructose, and/or mannose.

One or more antioxidants can optionally be included in the extender toprovide additional protection against cold shock. Exemplary antioxidantsinclude butylated hydroxytoluene (BHT), its derivatives, and the like.However, other antioxidants useful in the cryopreservation of cells canalso be employed in the extender, and the selection of a particularantioxidant for a specific application is within the level of skill inthe art in light of the guidance provided herein.

The extender can also contain a substance that facilitates spermcapacitation. A variety of capacitation facilitators are known in theart and any can be employed in the extender. Examples include enzymessuch as alpha amylase, beta amylase, beta glucuronidase, which can beused in combination, if desired.

Finally, the extender preferably includes an antibiotic, sincesubstantial bacterial growth can threaten sperm viability and increasethe risk of infection of the host in artificial insemination or in vitrofertilization procedures. Any of a variety of antibiotics useful in thecryopreservation of cells can also be employed in the extender. Theselection of a suitable antibiotic depends on the species from which thesperm was obtained, the procedures involved in obtaining and handlingthe sperm sample, and the specific microorganism(s) to be targeted.Exemplary antibiotics include tylosin, gentamicin, lincomycin,spectinomycin, linco-spectin (a combination of lincomycin andspectinomycin), penicillin, streptomycin, and ticarcillin, which can beused alone or in combination. However, one skilled in the art canreadily determine other antibiotics suitable for use in the extender.

Exemplary extenders are discussed in greater detail below and in theexamples.

The sperm concentration is typically lower in the selected sperm samplethan in the source sample, and, as indicated above, when FACS isemployed, the dilution is significant. A typical sort based on sex-typecan produce a sample containing sperm at 6×10⁵ cells/ml catch buffer. Assuch a low concentration is not optimal for storage (at least for mostspecies tested), the cryopreservation method of the invention generallyconcentrates the selected sperm sample.

Cooling the Selected Sperm Sample

The second step in the cryopreservation method entails cooling theselected sperm sample, typically, by reducing the temperature at acontrolled rate. Cooling too rapidly can cause cold shock, which canresult in a loss of membrane integrity and cell function. The severityof the effects of cold shock vary from species to species and depend onfactors such as the rate of cooling and the temperature range. Undersuitable controlled cooling conditions, the sperm are able to adapt tothermal effects. Example 2, among others, describes conditions forcooling bovine sperm, and determining suitable conditions for coolingsperm of other species is within the level of skill in the art.

In a preferred embodiment of the invention, the selected sperm sample iscooled typically from about 22° Celsius, to about 5° Celsius, andcooling is generally carried out over a period of about 60 minutes toabout 24 hours, preferably over a period of about 90 minutes to about240 minutes, and most preferably over a period of about 90 minutes toabout 120 minutes. Cooling can be accomplished by any convenient method,including simply placing the selected sperm sample in a 5° Celsiusenvironment.

Isolation of Sperm Cells from the Selected Sperm Sample

After initial extension of the selected sperm sample, sperm are isolatedfrom the sample using any sufficiently gentle isolation method thatprovides at least about 50% recovery of sperm, more preferably about 75%to about 90% recovery of sperm, and most preferably about 80% to about90% recovery of sperm. During the isolation step, the cooled spermshould generally be kept cold, i.e., between about 1 and about 8°Celsius, and preferably close to 4 or 5° Celsius.

Any of a variety of methods suitable for recovering cells from asuspension can be used to isolate the sperm, including for example,filtration, sedimentation, and centrifugation. In an exemplary,preferred embodiment, the selected sperm sample is aliquoted into 50 mltubes at volumes not exceeding about 27 ml, and preferably between about20 to about 27 ml. Centrifugation is carried out at about 4° Celsius, atabout 850×g, for about 20 minutes. Preferably, the centrifugation stepprovides at least about 50% to about 90% recovery of sperm, morepreferably about 60% to about 90% recovery of sperm, and most preferablyabout 70% to about 90% recovery of sperm. After isolation, thesupernatant is removed and the pellet is suspended by vortexing gentlyor repeated aspiration at 4° Celsius. The sperm concentration is thentypically determined (e.g., using a hemacytometer).

Final Extension of Isolated Sperm Cells

After isolation, the sperm are pooled, if desired, and extended withfinal extender to an appropriate concentration for freezing. Theconcentration of sperm after the final extension and prior to freezingis preferably in the range of about 1×10⁶/ml to about 300×10⁶/ml, morepreferably about 10×10⁶/ml to about 50×10⁶/ml, and most preferably about10×10⁶/ml to about 20×10⁶/ml.

The description of the initial extender above also applies to the finalextender, which can be the same as or different from the initialextender. In particular embodiments, the composition of the sperm sampleextended with the final extender is substantially similar to (if not thesame as) the composition of the sperm sample after addition of theinitial extender.

In a preferred embodiment of the invention, an egg yolk-Tris extender isused. After the addition of the extender, the sperm suspension comprisesglycerol (cryoprotectant); citric acid andTris[hydroxymethyl]aminomethane (buffer); egg yolk (organic substance);fructose (energy source); tylosin, gentamicin, and linco-spectin(antibiotics). The typical approximate concentrations of thesecomponents after addition of the final extender to the isolated spermare:

Components of Egg Yolk-Tris Extender Glycerol: 4-8% vol/vol Citric Acid:55-75 mM Tris [hydroxymethyl]aminomethane: 190-210 mM Egg yolk: 5-25%vol/vol Fructose: 45-65 mM Tylosin: 25-100 μg/ml Gentamicin: 200-300μg/ml Linco-spectin: 100-400 μg/ml* *100-400 μg/ml lincomycin and100-400 μg/ml spectinomycin

In a variation of this embodiment particularly suitable for freezingbovine sperm, the concentrations of these components after addition ofthe final extender to the isolated sperm are about 6% (vol/vol)glycerol, about 65 mM citric acid, about 200 mMTris[hydroxymethyl]aminomethane, about 20% (vol/vol) egg yolk, about 56mM fructose, about 50 μg/ml tylosin, about 250 gentamicin, and about150/300 μg/ml linco-spectin (i.e., 150 μg/ml lincomycin and 300 μg/mlspectinomycin), in deionized water.

In an alternative embodiment, an egg yolk-citrate extender is employed.After the addition of the extender, the sperm suspension comprisesglycerol (cryoprotectant); sodium citrate (buffer); egg yolk (organicsubstance); tylosin, gentamicin, and linco-spectin (antibiotics). Thetypical approximate concentrations of these components after addition ofthe final extender to the isolated sperm are:

Components of Egg Yolk-Citrate Extender Glycerol: 4-8% vol/vol SodiumCitrate: 60-80 mM Egg yolk: 5-25% vol/vol Tylosin: 25-100 μg/mlGentamicin: 200-300 μg/ml Linco-spectin: 100-400 μg/mL* *100-400 μg/mllincomycin and 100-400 μg/ml spectinomycin

Exemplary, preferred concentrations for freezing bovine sperm are about7% (vol/vol) glycerol, about 72 mM sodium citrate, about 20% (vol/vol)egg yolk, about 50 μg/ml tylosin, about 250 μg/ml gentamicin, and about250/300 μg/ml linco-spectin.

In another alternative embodiment, an egg yolk-TES-Tris (“EY TEST”)extender is employed. After the addition of the extender, the spermsuspension comprises glycerol (cryoprotectant); egg yolk and heatedmilk, e.g., homogenized milk containing 1.25% fructose with 10% glycerol(organic substances); tylosin, gentamicin, and linco-spectin(antibiotics). The typical approximate concentrations of thesecomponents after addition of the final extender to the isolated spermare:

Components of Egg Yolk TES-Tris Extender Glycerol: 3-7% vol/vol Tris[hydroxymethy-methyl]-2-aminoethanesulfonic 140-170 mM acid: Tris[hydroxymethyl]aminomethane; 60-80 mM Egg yolk: 5-25% vol/vol Fructose:5-12 mM Tylosin: 50-150 μg/ml Gentamicin: 400-600 μg/ml Linco-spectin:200-700 μg/mL* *200-700 μg/ml lincomycin and 200-700 μg/ml spectinomycin

Exemplary, preferred concentrations for freezing bovine sperm are about5% (vol/vol) glycerol, about 158 mMTris[hydroxymethy-methyl]-2-aminoethanesulfonic acid, about 72 mMTris[hydroxymethyl]aminomethane, about 20% (vol/vol) egg yolk, about 8mM fructose, about 100 μg/mL tylosin, about 500 μg/ml gentamicin, andabout 300/600 μg/ml linco-spectin.

In yet another alternative embodiment of the invention, a Milk extenderis employed. After the addition of the extender, the sperm suspensioncomprises glycerol (cryoprotectant); heated homogenized milk (organicsubstance); fructose (energy source); and tylosin, gentamicin, andlinco-spectin (antibiotics). The typical approximate concentrations ofthese components after addition of the final extender to the isolatedsperm are:

Components of Milk Extender Homogenized Milk 90% (Vol/Vol) Glycerol:3-7% (vol/vol) Fructose: 1.25% (wt/vol) Tylosin: 50 μg/ml Gentamicin:250 μg/ml Linco-spectin: 250/300 μg/ml* *250-300 μg/ml lincomycin and250-300 μg/ml spectinomycin

Exemplary preferred concentrations for freezing bovine sperm are about90% milk, about 10% (vol/vol) glycerol, about 1.25% fructose (wt/vol?),about 50 tylosin, about 250 μg/ml gentamicin, and about 250/300 μg/mllinco-spectin.

Other extenders standardly used to freeze sperm can also be employed asthe final extender in freezing selected sperm. A variety of extendersoptimized for use in freezing sperm from various species have beendescribed, and many are commercially available. Freezing extenders forequine sperm typically consist of milk, egg yolk, various sugars,electrolytes and a cryoprotectant. Exemplary freezing extenders aredescribed by Squires, E. L., et al., Cooled and Frozen Stallion SemenAnimal Reprod. and Biotechnology Laboratory, Bulletin No. 69, Chapter 8,“Seminal Extenders” pp. 49-51 (July, 1999).

Equilibration and Freezing of Sperm

Extension of the sperm sample produces a suspension of sperm, which isthen transferred into containers for freezing. If the sperm are intendedfor use in fertilization, the cells are conveniently aliquoted intoindividual doses sufficient to achieve fertilization. The required dosecan vary substantially from one species to the next and is eitherwell-known (e.g., for cattle and horses) or can readily be determined.In the case of sex-selected bovine sperm, convenient doses range fromabout 1.0×10⁶ sperm to about 3.0×10⁶ sperm.

Any suitable container can be employed for freezing, including, forexample, an ampule, a vial, and a straw. Sperm intended for AI aretypically frozen in straws (e.g., 0.25 ml or 0.50 ml straws) designedfor use with an insemination gun. Preferably, a bolus of extender isdrawn into the straw, followed, in sequence, by air, sperm, air, andextender, so that the sperm are flanked on either side by an air space,which separates the sperm from a bolus of extender at either end of thestraw.

Prior to freezing, the sperm are generally allowed to equilibrate atabout 5° C. Preferably, the sperm are allowed to equilibrate for aperiod in the range of about 1 hour to about 18 hours, more preferablybetween about 3 hours and about 18 hours, and most preferably betweenabout 3 hours and about 6 hours (see Example 2). Followingequilibration, any standard freezing method can be employed, providedthe freezing rate is not too rapid (i.e., not in excess of about 0.5°C./minute). Preferably, the freezing rate is about 0.5° C./minute. In anexemplary, preferred embodiment, the sperm are placed in static liquidnitrogen vapor, and freezing is carried out in three distinct stagesover a period of about 10 minutes. In the first stage of freezing, thesperm are cooled from about 5° C. to about −15° C. at a rate of about40° C./minute to about 65° C./minute. In the second stage of freezing,the sperm are cooled from about −15° C. to about −60° C. at a rate ofabout 25° C./minute to about 35° C./minute. In the third stage, thesperm are plunged into liquid nitrogen at about −100° C.

Selected Sperm Samples

In addition to a freezing method, the invention provides a frozen spermsample including sperm selected from a source sample for a particularcharacteristic. The sperm can be from any species, including any ofthose discussed above with reference to the freezing method. Theinvention encompasses frozen sperm selected for any characteristic byany suitable method, such as those described above. Preferredembodiments include frozen sex-selected human, bovine, equine, porcine,ovine, elk, or bison sperm. Sex-selection is preferably carried outusing flow cytometry as described generally above.

Also within the scope of the invention is a container containing afrozen sperm sample according to the invention. The container can beformed from any material that does not react with the frozen spermsample and can have any shape or other feature that facilitates use ofthe sample for the intended application. For samples intended for use inAI, for example, the container is conveniently a straw (e.g., 0.25 ml or0.5 ml straw) designed for use with an insemination gun. The containeris sealed in any manner suitable for preserving the sample at theintended storage temperature, which is typically below −80° Celsius.0.25 ml straws can be sealed, for instance, with PVC powder,ultrasonically, or with a cotton-polyvinyl plug and/or a stainless steelball (BB).

As the frozen sperm sample of the invention is typically thawed beforeuse, the invention also provides a thawed, previously frozen, selectedsperm sample and a container including such a thawed sample.

Methods of Using the Selected Sperm Sample

The frozen selected sperm sample of the invention is suitable for use inany method in which sperm are used. The sample can be thawed and used inany conventional fertilization method, such as artificial inseminationor in vitro fertilization. Thawing is carried out in the same manner asfor frozen, non-selected sperm. Briefly, the straw containing the frozensperm is submerged in a water bath maintained at a temperature of about35° C. to about 37° C. for a period of about 20 to about 30 seconds.After thawing, semen deposition (e.g., insemination) is carried outaccording to standard procedures, taking care to protect the sperm fromenvironmental fluctuations.

EXAMPLES Example 1 Effects of Dilution on Sperm Objective To Determinethe Effect of Sperm Concentration on Sperm Motility for Non-Frozen,Non-Sorted, but Highly Diluted Sperm A. Effects of Dilution onNon-Washed Sperm

1. Collection of Source Sample.

Sperm were collected from bulls on a routine collection schedule usingan artificial vagina as described in Schenk J., Proc 17th NAAB, p. 48-58(1998), and Saacke R G, Proc NAAB Tech Conf Al Reprod. 41:22-27 (1972).All ejaculates used contained greater than 50% progressively motile andgreater than 75% morphologically normal sperm. Antibiotics were added tothe raw ejaculate as described by Shin S., Proc NAAB Tech Conf AlReprod. 11:33-38 (1986) within 15 minutes of collection, and theconcentration of sperm was determined using a spectrophotometer.

2. Methods.

Sperm from 4 bulls were diluted to 1.25, 2.5, 5, 10, 15, and 20×10⁶/mlusing an egg yolk-citrate extender (EYC) prepared with 20% egg yolk(vol/vol) in 72 mM sodium citrate, 50 μg/ml tylosin, 250 μg/mlgentamicin, and 250/300 μg/ml linco-spectin. Each sample was prepared induplicate (2 tubes/dilution/bull) and comprised 8 ml total volume pertube. All samples were incubated for 60 minutes at 22° C., after whichthey were centrifuged using a swinging bucket centrifuge (Eppendorf,Model #5810R) at 600×g for 10 minutes to concentrate the sperm. Aftercentrifugation, the supernatant from one set of the duplicate tubes wasnot removed; the sperm were resuspended in the same medium and at theoriginal concentration by repeated gentle aspiration using a 5-mlserological pipette. (The second set of the duplicate tubes were used inExample IB.) Sperm samples were then cooled to 5° C. at 0.2° C./min over90 minutes. These sperm were termed “non-washed sperm.” All samples wereincubated at 5° C. for 24 or 48 h post-collection.

3. Evaluation of Motility.

After incubation, the samples were warmed to 37° C. using a dry blockincubator for 10 minutes prior to determination of motility. For thisexperiment, a single, blind estimate of the percentage of progressivelymotile sperm was determined for each sample. Progressive sperm motilitywas determined subjectively for each subclass by a single observer(×200, phase-contrast microscopy); another person prepared themicroscope slides in a randomized manner so the observer was unaware oftreatments.

4. Statistical Analysis.

Data were analyzed by analysis of variance (SAS Institute, Cary, N.C.)with factors bulls and initial dilution concentration. Separate analyseswere done for each incubation time. Dilution trends were tested using(log) linear contrasts.

5. Results.

Data for non-washed sperm (Table 1) revealed (log) linear relationships(P<0.01) for both incubation times. Percentages of motile spermincreased as sperm concentration increased from 1.25×10⁶/ml to10×10⁶/ml, but there was little difference thereafter. The cubic termwas significant (P<0.05) for 24-h and marginally significant (P<0.1) for48-h incubations. There was a bull effect (P<0.01) at both times.

TABLE 1 Effects of cooling on non-washed sperm motility (%) aftercooling to 5° C. Dilution Incubation at 5° C. (10⁶/ml) 24 h^(a) 48 h^(b)1.25 18^(c)  0^(c) 2.5 38^(c,d)  6^(c,d) 5.0 56^(d) 31^(d,e) 10.0 61^(d)42^(e) 15.0 55^(d) 44^(e) 20.0 58^(d) 41^(e) S.E^(.f)  5.6  6.4^(a)(log) linear (P < 0.01) and cubic effects (P < 0.05). ^(b)(log)linear (P < 0.01) and cubic effects (P < 0.1). ^(c,d,e)Means withincolumns without common superscripts differ (P < 0.05). ^(f){square rootover (error mean square of ANOVA)} ÷ {square root over (N)} (SASInstitute, Cary, NC, USA)

B. Effects of Dilution on Washed Sperm

1. Collection of Source Sample.

The second set of the duplicate tubes containing samples prepared inExample 1A were used in this experiment.

2. Methods.

The sperm were diluted, incubated and concentrated by centrifugation asin Example IA. Following centrifugation, 7.1 ml of the supernatant wasaspirated from each tube, removing most of the seminal plasma andleaving the sperm in a 900-μl pellet. The sperm were diluted with EYC(see Example 1A) to make 10×10⁶/ml or 20×10⁶/ml sperm suspensions. Thesamples were then cooled to 5° C. over 90 minutes as in Example 1A.

3. Evaluation of Motility.

The samples were warmed and evaluated for progressive motility as inExample 1A.

4. Statistical Analysis.

Data were analyzed as in Example 1A. In addition, data in Example 1Bwere analyzed for incubation concentration at 5° C.

5. Results.

Data for washed sperm (Table 2) revealed no significant treatmenteffects when sperm were evaluated after 24 h. However, after storage for48 h at 5° C., there were bull, initial dilution, incubationconcentration and bull by incubation effects (P<0.05). More spermremained motile when held at 20×10⁶/ml than at 10×10⁶/ml (31% vs. 20%;P<0.05). Initial dilutions of 1.25, 2.5, and 5×10⁶ sperm/ml resulted inlower progressive motility than 10×10⁶ sperm/ml (P<0.05), withrespective main effect means of 19, 20, 27, and 37% motile sperm.

TABLE 2 Cumulative effects of washing, dilution, concentration andcooling on progressive sperm motility (%) Sperm conc (10⁶/ml) during 1 hStorage at 5° C. - Sperm Concentration and Duration preincubation 24 h48 h^(a) at 37° C. 20 × 10⁶/ml 10 × 10⁶/ml 20 × 10⁶/ml 10 × 10⁶/ml^(b)1.25 45 49 24 15 2.5 51 40 29 11 5.0 54 54 32 21 10.0 51 50 40 34 15.060 41 20.0 55 40 ^(a)Concentration to 20 × 10⁶ sperm/ml was superior (P< 0.05) to 10 × 10⁶ sperm/ml after 48 h storage. Also, initial dilutionto 10 × 10⁶ was superior to lower dilutions (P < 0.05). Pooled standarderrors ({square root over (error mean square of ANOVA)} ÷ {square rootover (N)} were 4.0 for 24 h, and 2.8 for 48 h incubations.^(b)Significant (log) linear trend (P < 0.06).

C. Conclusion

High sperm dilution and cooling resulted in a substantial reduction inthe percentage of motile sperm, regardless of the presence or removal ofseminal plasma. However, this dilution effect was greatly attenuated byconcentrating the diluted sperm to 10×10⁶/ml and even more, to 20×10⁶/mlbefore storage at 5° C. Sperm from some bulls tolerated dilution betterthan sperm from other bulls; however, the bull differences found aretypical. Extremely dilute sperm might be compromised during sorting, inpart, by removal of protective compounds in seminal plasma.

Example 2 Effects of Equilibration Time Before Freezing Sorted SpermObjective To Evaluate the Effect of Equilibration Times (3, 6 and 18 h,5° C.) Before Freezing on Flow-Sorted Sperm

The following experiment was replicated in its entirety using the samebulls:

1. Collection of Source Sample.

Sperm of 4 bulls were collected and prepared as described in Example 1A.

2. Methods.

a) Staining and Preparation for Sort.

i) Preparation of Stain Stock Solution:

-   -   a stock solution of 8.89 mM Hoechst 33342 (bis-Benzimide        H-33342; #190305, ICN Biomedicals Inc., Aurora, Ohio) was        prepared in deionized water.

ii) Sperm Stain Procedure:

-   -   sperm were diluted in a modified TALP buffer (Table 3) to        400×10⁶ sperm/ml. Following dilution, Hoechst 33342 dye was        added to the sperm suspensions at a concentration of 224 μM.        After the stain was added to the sperm suspensions, the samples        were incubated for 60 minutes at 34° C. Following incubation,        sperm were diluted to 100×10⁶/ml with TALP containing 2.67%        clarified egg yolk and 0.002% food coloring dye (FD& C #40)        which quenches the fluorescence of Hoechst 33342 in sperm with        compromised cell membranes, allowing them to be gated out during        the sorting process. Just prior to flow sorting, samples were        filtered at unit gravity through a 40-μm nylon mesh filter to        remove any debris and/or clumped sperm.

b) Sorting.

-   -   A two-line argon laser operating at 351 and 364 nm and 150 mW        was used to excite the Hoechst 33342 dye. The flow        cytometer/cell sorter used was an SX MoFlo® (Cytomation, Inc.,        Fort Collins, Colo., USA) operating at 50 psi, A Tris-based        sheath fluid was used, consisting of        Tris(hydroxymethyl)aminomethane (Tris; 197.0 mM; #T-1503, Sigma        Chemical Co., St. Louis, Mo., USA), citric acid monohydrate        (55.4 mM; #C-7129, Sigma Chemical Co., St. Louis, Mo., USA) and        fructose (47.5 mM; #F-0127, Sigma Chemical Co., St. Louis, Mo.,        USA). Baseline antibiotics were also added to the Tris-based        sheath fluid consisting of 0.58 g/L of penicillin and 0.05 g/L        of streptomycin sulfate.

The sperm were sorted by a process referred to as “bulk sorting” whichpermits rapid accumulation of large numbers of sperm so that large-scaleexamples can be done within a reasonable time. The sperm pass throughthe flow cytometer under the standard operating conditions with theexception that all droplets containing viable sperm were collected intoa single tube rather than being sorted into 2 tubes based upon specificcharacteristics (e.g., sorting by sex-type). Sperm were sorted on thebasis of viability; hence, sperm that have compromised plasma membraneswere excluded during bulk sorting.

Stained sperm were maintained at 22±1° C. during sorting. Bulk sortedsperm were collected in 50-ml plastic tubes containing 2 ml of 20% eggyolk-Tris extender prepared with 20% egg yolk (vol/vol) in 200 mM Tris,65 mM citric acid, 56 mM fructose, 50 μg/ml tylosin, 250 μg/mlgentamicin, and 150/300 μg/ml linco-spectin in deionized water. The eggyolk-Tris extender was termed “Tris-A fraction” to denote the lack ofglycerol at this point in the procedure. Sperm were collected in tubesto contain 12 ml and approximately 6×10⁶ sperm. The sperm weresubsequently incubated at 22° C. for 1 to 3 h to simulate conditions ofa sort based on sex-type.

c) Preparation for Freezing.

-   -   Following incubation, the sorted sperm were cooled to 5° C. over        the period of 70 minutes. After cooling, the contents of the two        tubes were pooled and transferred to a refrigerated, swinging        bucket centrifuge set at 5° C. and centrifuged at 850×g for 20        minutes. After removing the supernatant, processing continued at        5° C. by adding about 150 μl of Tris-A fraction extender to        about 150-μl of sperm pellet to bring the sperm concentration to        approximately 40×10⁶/ml. The sperm of individual bulls were        pooled and diluted immediately with an equal volume of egg        yolk-Tris extender containing 12% (v/v) glycerol (“Tris-B        fraction”). The Tris-B fraction was added to the sperm        suspension as 2 equal volumes at 15-minute intervals to adjust        the final sperm concentration to 20×10⁶/ml. The final glycerol        concentration of the complete egg yolk-Tris extender containing        the sperm was 6% (v/v).

d) Equilibration and Freezing.

-   -   Extended sperm were then packaged into 0.25-ml polyvinylchloride        straws to be frozen by routine procedures on racks in static        liquid nitrogen vapor. Two straws from each of 4 bulls were        frozen after 3, 6 and 18 h of total equilibration time at 5° C.

3. Evaluation of Post-Thaw Motility.

Straws were thawed in a 37° C. water bath for 30 sec. Blind estimates ofprogressive motility were made after incubating samples at 37° C. for 0,1 and 2 h post-thawing. Each of two observers estimated progressivesperm motility from each of two straws of semen. These four blindestimates for each experimental unit represent subsampling.

4. Statistical Analysis.

Statistically, the subsamples were analyzed as a subplot to the mainplot least-squares ANOVAs to analyze effects of any observer andobserver x treatment interaction. N refers to the number of experimentalunits, not subsamples; standard errors were calculated on the basis ofmeans of the 4 subsamples from error mean squares of ANOVAs and thenumbers of experimental units; least-squares means are presented.

Treatment effects were evaluated via separate ANOVAs for each incubationtime. The model included bulls as a random effect and equilibration timeand observer as fixed effects; the subplot consisted of the observerterm and related interactions.

5. Results.

The 3- or 6-h equilibration times were superior to 18-h (Table 4), basedon the percentage of progressively motile sperm, for 0 and 1 h (P<0.01)but not 2 h of post-thaw incubation. Effects of bull were evident at 1and 2 h incubation times (P<0.05), but not at 0 h. There was nosignificant (P>0.1) bull by equilibration time interaction nor was therea significant observer effect for any response.

TABLE 3 Modified TALP Buffer Ingredient Concentration NaCl 95.0 mM KCl3.0 mM NaHPO₄ 0.3 mM NaHCO₃ 10.0 mM MgCl₂•6H₂O 0.4 mM Na Pyruvate 2.0 mMGlucose 5.0 mM Na lactate 25.0 mM HEPES^(a) 40.0 mM Bovine serumalbumin^(b) 3.0 mg/ml Gentamycin Sulfate 30.0 μg/ml ^(a)#H3375, SigmaChemical Co., St. Louis, MO, USA ^(b)#US70195, fraction V; Amersham/LifeScience, Cleveland, OH, USA

TABLE 4 Effect of pre-freeze equilibration time on post-thaw progressivemotility (%) Equilibration Post-thaw incubation at 37° C. at 5° C. 0 h 1h 2 h 3 h 41^(a) 36^(a,b) 16 6 h 41^(a) 37^(a) 18 18 h  35^(b) 31^(b) 12S.E.^(c)  1.5  0.8 2.0 ^(a,b)Within columns, means without commonsuperscripts differ (P < 0.05), Tukey's HSD. ^(c)Pooled standard errors,{square root over (error mean square of ANOVA)} ÷ {square root over (N)}

6. Conclusion.

The results indicated no differences in post-thaw sperm motility between3 and 6 h of total equilibration time at 5° C., but there was asignificant decline in sperm motility following 18 h of equilibration at5° C. before freezing. The 3- to 6-h range permits pooling 2 consecutive3-h sorting batches for freezing sperm without decreasing post-thawmotility.

As the bull by equilibration-time interaction was not significant, 3 to6 h equilibration was adequate, with the caveat that only 4 bulls wereused. The optimum equilibration time for a minority of bulls is expectedto be >6 h.

Example 3 Effects of Stain Concentration and Laser Power on Sorted SpermObjective To Evaluate the Effects of Hoechst 33342 Dye Concentration inCombination with Laser Intensity on Flow-Sorted Sperm

1. Collection of Source Sample.

Sperm of 6 bulls were collected and prepared as described in Example 1A.

2. Methods.

a) Experimental Design.

-   -   One ejaculate (2 bulls) and 2 ejaculates on different days (4        bulls) were used in a 2 by 2 design plus control.

b) Staining and Sorting.

-   -   Staining, preparation for sorting and sorting sperm were        achieved as described in Example 2 except that the Hoechst 33342        dye was added to sperm suspensions at a final concentration of        149 μM or 224 μM; and sperm were bulked-sorted with the laser        operating at 100 mW or 150 mW of incident power. Bulk-sorted        sperm were collected into 50-ml plastic tubes as described in        Example 2. Four tubes containing approximately 15×10⁶ total        sperm/tube were collected over 1 h for each bull. The sorted        sperm were incubated for 1 h at 22° C. to simulate a longer        sorting time.

c) Preparation for Freezing.

-   -   Following incubation, the sperm were cooled as in Example 2. The        sperm were then concentrated by centrifugation at 5° C. at 850×g        for 20 minutes. After removing the supernatant, 150 μl of Tris-A        fraction extender was added to each 150-μl sperm pellet at 5° C.        All of the sperm pellets were suspended by gentle repeated        aspiration and the sperm of individual bulls were pooled. Tris        B-fraction extender was added stepwise as described in        Example 2. A non-stained, non-sorted control for each bull was        prepared at 20×10⁶ sperm/ml in Tris extender containing 6%        glycerol and cooled to 5° C. while the bulk-sorted sperm were        being prepared.

d) Equilibration and Freezing.

-   -   The control and sorted sperm were packaged into 0.25-ml        polyvinylchloride straws as described in Example 2, equilibrated        at 5° C. for 3 h and then frozen conventionally.

3. Evaluation of Post-Thaw Motility.

Straws were thawed and evaluated as described in Example 2.

4. Statistical Analysis.

A general description of statistical analyses is provided in Example 2.Specifically, treatment effects were evaluated via ANOVA. The modelincluded dye concentration, laser intensity and bulls in the main plot,and observer and related interactions in the subplot. Bulls wereconsidered a random effect and the other factors as fixed.

5. Results.

Bull effects were significant for percentages of progressively motilesperm immediately after thawing (P<0.1) and after 1 h and 2 h ofincubation at 37° C. (P<0.05). There was no effect of dye concentrationor bull by dye concentration on sperm motility at any incubation time.With bulls considered as a random effect, 150 mW of laser power resultedin lower post-thaw motility of sperm than 100 mW at 0 h of incubation(P<0.1), but not at other incubation times (Table 5). If bulls areconsidered as fixed effects, 150 mW of power resulted in lower spermmotility than 100 mW (P<0.05) at all 3 incubation times. There was aneffect of bull by laser power (P<0.05) on sperm motility at 1 h, but notat 0-h or 2-h incubation times. Also, the higher laser power resulted inlower sperm motility than the control (P<0.05) at 0- and 1-h incubationtimes (Table 5). There was a significant observer effect at 1-h, but notat 0-h or 2-h, incubation times. There was no observer by treatmentinteraction (P>0.1).

TABLE 5 Effects of laser intensity and dye concentration on post-thawmotility (%). Incubation at 37° C. 0 h 1 h 2 h Main effect means Control49 44 33 Dye Concentration 149 μM 41 39 30 224 μM 42 39 30 LaserIntensity 100 mW 46 42 33 150 mW   38^(a)  35^(b) 27 S.E.^(c)   2.2  1.2 1.3 ^(a)Significant main effect (P < 0.1) and differs from control(P < 0.05). ^(b)Differs from control (P < 0.05). ^(c)Pooled standarderrors, {square root over (error mean square of ANOVA)} ÷ {square rootover (N)}

6. Conclusion.

Percentages of progressively motile sperm post-thaw were diminished bythe staining and sorting process. Higher laser intensity was moredamaging than the lower laser intensity. There was no effect of dyeconcentration on post-thaw sperm motility. Thus, excitation of thesperm-bound Hoechst 33342 dye at lower laser intensities is lessdamaging and that staining sperm at the higher dye concentration had nodetrimental effect on post-thaw motility. The damage observed waspresumably to the sperm-motility apparatus.

Example 4 Evaluation of Pre-Sort Staining Procedures and Selection ofExtenders for the Cryopreservation of Sperm Objective (1) To EvaluateThree Pre-Sort Treatments for Sperm; and, (2) and to Evaluate SheathFluid and Extender Combinations for the Cryopreservation of Flow-SortedSperm

The following experiment was replicated in its entirety:

1. Collection of Source Sample.

Sperm from 4 bulls were collected and prepared as described in Example1A.

2. Methods.

a) Experimental Design.

-   -   A 3 (pre-sort treatments) by 3 (extenders) by 2 (sheath fluids)        by 4 (bulls) by 2 (observers) factorial experiment was designed        to determine the best procedure to hold sperm prior to sorting,        and to evaluate three extenders for cryopreserving the sorted        sperm.

b) Sample Preparation and Staining.

-   -   Freshly collected sperm from each of 4 bulls were treated as        follows:        -   (1) diluted to 400×10⁶/ml in modified TALP (see Example 2,            Table 3) and stained for 1 h at 34° C. before bulk-sorting            (“Dilute—0 h”);        -   (2) incubated neat at 22° C. for 3 h before dilution,            staining and sorting (“Neat—3 h”); or,        -   (3) diluted and stained as “Dilute—0 h and then incubated at            22° C. for 3 h before bulk-sorting (“Diluted—3 h”).

c) Extenders.

-   -   The following freezing extenders were compared: EYC (see        Example 1) containing 7% glycerol, egg yolk-Tris (see Example 2)        containing 6% glycerol, and egg yolk-TES-Tris (TEST) containing        5% glycerol. EYC “A Fraction” refers to the EYC extender        containing no glycerol, and EYC “B Fraction” refers to EYC        extender containing twice the final, desired glycerol        concentration (i.e., 14%). Thus, when EYC A and B fractions are        combined in equal volume, the final EYC extender contains 7%        glycerol. Tris A and B fractions are similarly named, and        described in Example 2. TEST extender is prepared as a complete        extender containing 5% glycerol; hence, there were no “A” and        “B” fractions for TEST.

d) Sheath Fluid.

-   -   Sheath fluid was either 98.6 mM sodium citrate dihydrate        (#S279-3, Fisher Scientific, Fair Lawn, N.J.) or Tris as        described in Example 2. Both types of sheath fluid were adjusted        to pH 6.8; osmolality was about 270 to 280 mOsm/kg. Tris sheath        fluid was used to collect sperm that were later extended in egg        yolk-Tris and TEST freezing extenders. Sheath fluid containing        98.6 mM sodium citrate dihydrate was used to collect sperm to        later be extended in EYC freezing extender.

e) Sorting.

-   -   Approximately 58×10⁶ sperm for each combination of pre-sort        treatment, sheath fluid and extender were bulk-sorted as        described in Example 2 using 150 mW of incident laser power. For        each sort, sperm were collected over approximately 1 h. After        sorting, the samples were incubated at 22° C. for 2 h to        simulate a 3 h sort.

f) Preparation for Freezing.

-   -   Following incubation, the sperm were cooled as described in        Example 2. After cooling, the samples were centrifuged at 5° C.        at 850×g for 20 min. Each sample comprised about 28 ml total        volume and was contained in a 50-ml plastic tube    -   After the supernatant was removed, the sperm were returned to a        5° C. cold room for extension. Samples were extended to        40×10⁶/ml by depositing 131 μl of the sperm suspension into 69        μl of A-fraction EYC, A-fraction egg yolk Tris, or TEST        extender. Immediately, suspensions were adjusted to 20×10⁶        sperm/ml with the addition of the matched glycerol containing        extender (i.e., B-fraction EYC, B-fraction Tris) or TEST.        B-fraction extenders were added to their respective samples        stepwise (2×) at 15-min intervals as described in Example 2. The        TEST was added to sperm stepwise in the same manner as B        fraction EYC and Tris extenders.

g) Equilibration and Freezing.

-   -   Sperm were packaged into 0.25-ml polyvinylchloride straws,        equilibrated for 3 h at 5° C. and then frozen in static liquid        nitrogen vapor.

3. Evaluation of Post-Thaw Motility.

Thawing and post-thaw evaluations of sperm were done as described forExample 2.

4. Statistical Analysis.

A general description of statistical analyses is provided in Example 2.Specifically, treatment effects were evaluated via separate analyses ofvariance for each post-thaw incubation time. The main plot includedpre-sort treatment, extenders, and bulls; the subplot consisted ofobservers and associated interactions. Bulls were considered a randomeffect, and the other factors, fixed. The entire experiment wasreplicated twice. Tukey's HSD test was used to separate means.

5. Results.

Post-thaw progressive motility of bulk-sorted sperm was affected(P<0.05) by extender and bulls at each post-thaw incubation time and bypre-sort procedure at 0 h of incubation (Table 6). There were nodifferences due to sheath fluids (P>0.05). At 0-h post-thaw incubation,use of the neat—3 h treatment resulted in more motile sperm afterfreezing and thawing than the other 2 pre-sort staining treatments(P<0.05; Table 6). However, pre-sort procedures were not statisticallysignificant after post-thaw incubation of sperm for 1 or 2 h with bullsconsidered as a random effect. Importantly, at these 2 incubation times,there were significant pre-sort treatment by bull interactions (P<0.05).Furthermore, pre-sort treatment would have been a significant effect atall post-thaw incubation times had bulls been considered as fixedeffects.

Immediately after thawing (0 h), TEST was the best extender, but after 1or 2 h of incubation of 37° C., Tris was the best extender. Importantly,there was no pre-sort treatment by extender interaction for anyresponse. There were observer effects (P<0.01) at all incubation times,but no observer by treatment interactions. There was a bull by extenderinteraction (P<0.05) at all 3 incubation times.

TABLE 6 Main effects of pre-sort treatment and freezing extenders onpost-thaw progressive motility (%) Incubation at 37° C. Pre-sortprocedure Extender 0 h 1 h 2 h Dilute - 0 h Mean 39^(a) 32 22 Neat - 3 hMean 43^(b) 36 25 Dilute - 3 h Mean 38^(a) 31 19 Mean EYC 36^(a) 29^(a)17^(a) Mean Tris 40^(b) 39^(b) 29^(b) Mean TEST 44^(c) 33^(c) 20^(a)S.E.^(d)  0.8  0.8  0.7 ^(a,b,c)Means within columns, within maineffects, without common superscripts differ (P < 0.05). ^(d)Pooledstandard errors {square root over (error mean square of ANOVA)} ÷{square root over (N)}

6. Conclusion.

This study showed that holding sperm neat for 3 h before dilution,staining and sorting was better than immediate dilution and staining 0 hor 3 h later. Thus, by 3 h into the sort, it is best to continue with anew aliquot of the original ejaculate that was held neat 3 h and thenstained, rather than continuing with the original sample of spermstained and held at 400×10⁶ sperm/ml.

Even though TEST extender provided higher pott-thaw motility at 0 h,Tris was the superior extender when sperm were stressed by incubation at37° C. Either sheath fluid worked equally well for each extender. Basedon these results, we have incorporated the use of Tris sheath fluid incombination with Tris freezing extender into our standard operatingprocedure.

Example 5 Effects of Extender Additives on Sorted Sperm Objective ToEvaluate the Effect of Adding Sodium Dodecyl Sulfate (“SDS”) to theFreezing Extender on Flow-Sorted Sperm A. Evaluation of Effect ofConcentration of SDS in Freezing Extender

1. Collection of Source Sample.

Sperm of 6 bulls were collected and prepared as described in Example 1A.

2. Methods.

Sperm from each of 6 bulls were extended to 20×10⁶/ml in 20% whole eggTris (“WET”) extender containing 0, 0.03, 0.06, 0.09, or 0.12 percentSDS, packaged into straws and frozen. WET extender was prepared using3.028 g of Tris[hydroxymethyl]aminomethane, 1.78 g of citric acidmonohydrate, and 1.25 g of fructose per 100 ml of double distilledwater, to which 20% whole egg (vol/vol) was added. The WET extender wasprepared at a pH of about 7.0 and contained a final glycerolconcentration of about 6% (vol/vol). The WET extender also contained1000 IU of penicillin “G” sodium and 100 μg of streptomycin sulfate/ml.

3. Results.

The respective means (n=1 sample from each of 6 bulls) were 51, 51, 50,51, and 48% progressive motile sperm approximately 10 minutes post-thaw.Based on these results, 0.06 percent SDS was used in Example 5B.

B. Evaluation of the Effects of 0.06 Percent SDS in Various FreezingExtenders on Post-Thaw Motility of Flow-Sorted Sperm

1. Collection of Source Sample.

Sperm of 8 bulls were collected and prepared as described in Example 1A.

2. Methods.

Post-thaw motility was studied for sperm frozen in egg yolk—Tris (seeExample 2) and WET extenders (see Example 5A) with and without 0.06%SDS. Final glycerol content for both extenders was 6%.

a) Staining, Preparation for Sort, Sorting.

-   -   Stained sperm samples were prepared from an ejaculate from each        of 8 bulls as described in Example 2. Stained sperm were        bulked-sorted using Tris sheath fluid as described in Example 2        except that the sort was achieved using 135 mW of incident laser        power. Sorted sperm were collected in a 50-ml plastic tube        containing 2 ml of A-fraction freezing buffer for each extender;        15×10⁶ total sorted sperm (25 ml) for each treatment were        collected and incubated for 1 h at 22° C. to simulate longer        sorting.

b) Preparation for Freezing.

-   -   Diluted sperm were then cooled to 5° C. over minutes. An equal        volume of appropriate B-fraction extender was added stepwise        (2×) at 15-minute intervals to each 50-ml plastic tube        containing sorted sperm. Aliquots, of 25 ml/extender treatment        were concentrated by centrifugation for 20 minutes at 850×g in a        refrigerated centrifuge. The supernatant was removed leaving a        600 μL, sperm pellet, which was suspended by gentle vortexing        for 15 seconds. No additional extender was added to the sperm        pellet since the suspension containing the pellet already        contained glycerol. The concentration of the sperm suspension        was approximately 20×10⁶/ml. A non-stained, non-sorted control        for each bull was prepared at 20×10⁶ sperm/ml in egg-yolk-Tris        extender containing 6% glycerol. The control was placed in a        5° C. cold room while bulk-sorting occurred.

c) Equilibration and Freezing.

-   -   All control and bulk-sorted sperm were packaged and frozen at        the same time. Sperm were packaged into 0.25-ml        polyvinylchloride straws, equilibrated for about 3 h to about 6        h at 5° C. and then frozen in static liquid nitrogen vapor.

3. Evaluation of Post-Thaw Motility.

Thawing and post-thaw evaluations of sperm were done as described forExample 2 with the exception that progressive motility was evaluated 0.5and 2.0 h after incubation.

4. Statistical Analysis.

A general description of statistical analyses is provided in Example 2.Specifically, treatment effects were evaluated via separate analyses ofvariance for each incubation time; the model included bull and extenderin the main plot and observer and related interactions in the subplot.Differences in means were determined by the least significant differencetest.

5. Results.

Extender affected (P<0.05) progressive motility of sperm after 0.5 or 2h post-thaw incubation (Table 7). At 0.5 h, WET plus SDS resulted inlower motility than Tris with SDS. At 2 h, all treatments withbulk-sorted sperm were worse than the non-sorted control sperm. Therewere significant bull and observer effects (P<0.01) at both incubationtimes, but no observer by treatment interactions.

TABLE 7 Effect of extender on post-thaw progressive motility (%)Incubation at 37° C. Extender 0.5 h 2 h Tris (non-sort) 42^(a) 41^(a)Tris w/o SDS 40^(a,b) 35^(b) Tris w/SDS 42^(a) 37^(b) WET w/o SDS40^(a,b) 35^(b) WET w/SDS 38^(b) 35^(b) S.E.^(c)  1.0  1.2 ^(a,b)Meanswithin columns without common superscripts differ (P < 0.05).^(c){square root over (error mean square of ANOVA)} ÷ {square root over(N)}

6. Conclusion.

The inclusion of SDS in Tris or WET extenders did not benefit spermquality as determined by visual estimates of motility after thawing.Also, results using WET and Tris extenders were similar; hence, WETappeared as efficacious as Tris for cryopreserving sorted bovine sperm.

Example 6 Quality of Sperm Sexed by Flow Sorting for Field TrialsObjective To Evaluate Post-Thaw Quality of Sorted Sperm Based onAcrosomal Integrity

1. Collection of Source Sample.

Sperm of 3 bulls were collected and prepared as described in Example 1A.

2. Methods.

Sorted and non-sorted control sperm from the same ejaculate werestained, processed, and sorted as described in Example 2 except thesperm were sorted for sex-type at a 90% purity level. Sorted sperm werecollected to a volume of approximately 20 ml and were cooled to 5° C.for 90 minutes (0.2° C./min). After cooling, an equal volume of eggyolk-Tris B extender (see Example 2) was added to the sorted sperm in 2equal volumes at 15-minute intervals. Centrifugation and aspiration ofthe supernatant were achieved as described in Example 5. Aftercentrifugation and aspiration, egg yolk-Tris extender containing 6%glycerol (v/v) was added to the sperm pellet to bring the concentrationof sperm to about 20×10⁶/ml. Freezing and thawing were done as describedin Example 2 except that equilibration time was about 3 h.

3. Evaluation of Post-Thaw Motility.

Visual estimates of the percentage of progressively motile sperm at 37°C. were made approximately 10 minutes after thawing. The acrosomalintegrity of sperm was assessed using differential interference-contrastmicroscopy (×1000) after 2 h of incubation at 37° C. Sperm were treatedwith 40 mM sodium fluoride, a wet was smear made, and 100 sperm pertreatment were examined. Acrosomes were classified as: (a) intactacrosome, (b) swollen or damaged acrosome, or (c) missing acrosome(non-intact).

4. Statistical Analysis.

The data analyzed were from 19 different freeze dates balanced across 3bulls used in field trials. Treatment effects (sort vs. control) wereevaluated via analysis of variance with bulls as a fixed effect.

5. Results.

The percentage of progressively motile sperm post-thaw was significantlyhigher (P<0.05) for non-sorted sperm (50%) than for sorted sperm (46%;Table 8), despite removal of dead sperm during sorting. However, thepercentage of sperm with an intact acrosome was not different. Sortingincreased the percentage of sperm missing an acrosome, but also reducedthe percentage of sperm with a damaged acrosome, relative to controlsperm (P<0.05). There were significant differences among bulls forpercent of intact acrosomes (P<0.05), percent of non-intact acrosomes(P<0.01), and post-thaw progressive motility (P<0.01). There was a bullby sorting effect for post-thaw motility (p<0.01) but not for the otherresponses. From bulls A and B, differences in post-thaw motility betweensorted and unsorted sperm were near zero; for bull C, sorted sperm were10 percentage points (19%) lower in motility than control sperm.

TABLE 8 Effect of sorting on post-thaw motility (%) and acrosomal status(%) Acrosomal status Intact Damaged Non-intact Post-thaw motilityControl 64^(a) 20^(a) 15^(a) 50^(a) Sorted 65^(a) 14^(b) 21^(b) 46^(b)^(a,b)Column means with different superscripts differ (P < 0.05).

6. Conclusion.

Visual estimates of progressive motility for sorted, frozen sperm onaverage were slightly lower (4 percentage points; 8%) than for controlsperm, although this difference was larger for one bull. Theseevaluations were made approximately 10 minutes after thawing. The smallaverage difference is consistent with that for non-intact acrosomesafter 2 h of incubation. Sperm with a damaged or missing acrosome arelikely to be immotile. The increased percentage of sperm with anon-intact acrosome, for sorted samples, indicates damage associatedwith sorting or with cryopreservation before or after actual sorting.Presumably, sorting converted damaged acrosomes to missing acrosomes.Based on standard procedures for evaluation of sperm quality, there isno basis for assuming that fertilizing potential of these flow-sortedsperm should be severely compromised for most bulls.

Example 7 Sex-Selection and Cryopreservation of Bull Sperm Using 20% EggYolk-Tris Extender Objective To Provide a Protocol for theCryopreservation of Flow-Sorted Bull Sperm

1. Collection and Ejaculate Assessment.

Collect and prepare ejaculates as described in Example 1A. Selectejaculates from those bulls with >75% morphologically normal sperm.Visually estimate the percentage of progressively motile sperm(ejaculates that have progressive motility >60% are best for sorting).Add antibiotics to raw semen as follows: tylosin at a finalconcentration 100 μg/ml, gentamicin at a final concentration of 500μg/ml, and linco-spectin at a final concentration of 300/600 μg/ml.

2. Staining and Preparation for Sort.

Following the addition of the antibiotics to the raw semen sample, allow15-20 minutes before staining. Stain samples as described in Example 2.

3. Sorting.

Sort for both X- and Y-type sperm, setting the sorting gates for 90%purity. Sort sperm into 50-ml Falcon tubes containing 2 ml 20% eggyolk-Tris A-fraction extender (see Example 2) until each tube contains amaximum of 20 ml total volume (or a maximum of 2 h per sort) and finalsorted sperm concentration is 6×10⁵/ml. Note that additional 20% eggyolk-Tris-A fraction catch buffer must be added after the sort and priorto cooling so that the final percentage of egg yolk is at least 3%.

4. Preparation for Freezing.

Following the sort, cool the sorted samples to 5° C. over a period of 90minutes. After cooling, add 20% egg yolk-Tris B-fraction extender (seeExample 2) stepwise (2×) at 15 minutes intervals. The final volume ofTris B-fraction extender added to the sperm sample should be equal tothe volume of Tris A-fraction extender. The total volume of sperm sampleafter the Tris B-fraction extender is added should not exceed 27 mltotal volume.

After the Tris B-fraction extender is added to the sperm sample,concentrate the sample by centrifugation for 20 minutes at 850×g.Aspirate the supernatant leaving approximately 150 μl sperm pellet.Resuspend the sperm and pool the sperm for each individual bull.

5. Freezing.

Add complete egg yolk-Tris extender (6% glycerol) to achieve a finalsperm concentration of 20×10⁶/ml. Package the extended sperm into0.25-ml polyvinylchloride straws for freezing as described in Example 2.

Example 8 Evaluation of the Fertility of Flow-Sorted, Frozen Bull Spermin Field Studies Materials and Methods Semen Collection and Processing

Semen from young bulls of unknown fertility was collected via artificialvagina (see Example 1A). After determining sperm concentration with aspectrophotometer and subjective evaluation of progressive spermmotility, semen was processed and sorted as described in Example 2except that the sperm were sorted by sex-type at 90% purity using alaser incident power of about 135 to about 150 mW. Processing andfreezing was achieved as in Example 2 except that the equilibration timewas about 3 h. Cornell Universal Extender (Seidel G E Jr.,Theriogenology 1997; 48:1255-1264) was used for liquid semen in fieldtrials 1, 2, and 3. For frozen semen in field trials 2 and 3, theextender used was 2.9% Na citrate+20% egg yolk with a final glycerolconcentration of 7% (see Example 1). For field trials 4 through 11,sperm were frozen in a Tris-based extender composed of 200 mM Tris, 65mM citric acid, 56 mM fructose, 20% egg yolk, and a final glycerolconcentration of 6% (see Example 2). The sheath fluid used in the flowcytometer was 2.9% Na citrate (see Example 4) for trials 1, 2, and 3,and a Tris buffer for the remaining trials (see Example 2).

Sperm were packaged in 0.25-mi French straws in columns as small as 50μl in the center of the straw. To minimize dilution effects, low volumeswere used so there were at least 10⁷ sperm/ml. In most trials, a columnof extender without sperm was aspirated into the straw first to wet thecotton plug, followed by a small column of air, and then the sexedsperm. When sperm were frozen, one straw from each batch was thawed in35° C. water for 30 sec for quality control, and batches with less than25% progressive motility post thaw were discarded. A sample of sexedsperm from each batch was sonicated and analyzed by flow cytometry todetermine the accuracy of sexing.

Heifer Management and Artificial Insemination

The heifers used were in 6 widely scattered production units withdifferent management practices. Seasonal and breed differencescontributed further to the heterogeneity of the experiments (Table 9).Insofar as possible, treatments and controls were alternatedsystematically within bulls within inseminators as heifers entered theinsemination facilities.

Estrus was synchronized in one of 4 ways (Table 9): (1) 500 mg ofmelengesterol acetate (MGA) fed daily in 2.3 kg of grain for 14 daysfollowed by an i.m. injection of 25 mg prostaglandin F₂α (Lutalyse,Upjohn, Kalamazoo, Mich., USA) 17, 18 or 19 days after the last day offeeding MGA (MGA/PG); (2) a single injection of 25 mg of prostaglandinF₂α(PG); (3) 20 or 25 mg of prostaglandin F₂α injected i.m. at 12-dayintervals (PG/PG) or (4) 50 or μg of GnRH injected i.m., followed by 25mg of prostaglandin F₂α 7 days later (GnRH/PG).

Heifers were inspected visually for standing estrus mornings andevenings, but inseminated only in the evenings after 16:00,approximately ½ or 1 day after onset of estrus. Insemination was eitherinto the uterine body conventionally, or half into each uterine hornusing atraumatic embryo transfer sheaths (IMV, Minneapolis, Minn., USA).In the latter case, semen was deposited past the greater curvature ofthe uterine horn as far anterior as could be accomplished withouttrauma, identically to nonsurgical embryo transfer. In most cases, semenwas deposited between the anterior third and mid-cornua.

Most experiments included a frozen sperm control inseminated into theuterine body with 20 or 40×10⁶ sperm/dose from the same bulls used forsperm sorted for sex-type (“sexed”). This control served as a compositeestimate of the intrinsic, normal fertility of the heifers under thespecific field-trial conditions as well as the fertility of the bullsused and the skills of the inseminators. Some trials also included alow-dose, unsexed control group. Sometimes numbers of controlinseminations were planned to be ½ or ⅔ the number used for eachtreatment to obtain more information on sexed sperm. Frozen sexed andcontrol sperm were thawed for 20 to 30 sec in a 35 to 37° C. water bath.Various other details are summarized in Table 9.

Pregnancy was diagnosed by ultrasound 28 to 37 d post inseminationand/or 56 to 92 d post-insemination, at which time fetal sex wasdetermined in most trials, as described in Curran, S., Theriogenology1991; 36:809-814, without the operator's knowing insemination treatmentsof controls. Sexes of calves born were nearly identical to the fetal-sexdiagnosis. Data were analyzed by single-degree-of-freedom Chi squarecorrected for continuity; 2-tail tests were used unless 1-tail isspecified. Fewer than 5% of the inseminations were culled due to errorsof insemination treatment, frank infection of the reproductive tract,failure to traverse the cervix, etc. Decisions to cull animals fromexperiments were made shortly after insemination and were never based onthe pregnancy diagnosis.

TABLE 9 Procedural details of field trials Insemination Breeds of EstrusTrial dates heifers Bulls used Inseminators synchronization Comments 1May 20-23, 1997 Angus Nl, N2, AN4 A, B MGA/PG Included low- dosecontrols 2 Feb. 18-May 22, Angus N3, N4, N5, C, D PG/PG Low dose but no1998 crossbred N6 normal-dose controls; some heifers pregnant andaborted when synchronized 3 Jun. 2-Jun. 5, Angus AN4, AN5, N7, N8 B, DMGA/PG 1998 4 Feb. 10-13, Holstein J2, J4 C, D PG Very severe 1999 mud,snow, wind, and cold, driving rain 5 Feb. 24-26, Holstein J2, J4, J5 C,B, D PG/PG 1999 6 Apr. 14-16, Holstein J2, J3, J4, J5 C, D PG Someheifers 1999 were reproductive culls 7 Apr. 27-May 1, Hereford& AN1, AN4C MGA/PG Semen for 1 bull 1999 Angus shipped 6 h crossbred beforesorting; severe weather 8 Apr. 21-May 1, Angus H1, H2 E MGA/PG Feedlotheifers 1999 crossbred 9 May 5-8, 1999 Red Angus AR1, AR2 C, F MGA/PG 10May 31-Jun. 2, Angus AN4, AN7, AN8 B, D GnRH/PG 1999 11 Jul. 28-30,Holstein H2, H3 C, D PG/PG First replicate 1999 available in a muchlarger trail

RESULTS AND DISCUSSION

The data presented are from 11 consecutive, heterogeneous field trials,constrained by logistical aspects of the studies, such as having tomatch bulls to genetic needs of the herds, unavailability of fertilityinformation on bulls, limited numbers of heifers, unavailability of thesame inseminators across trials, severe weather in some trials, limitedamounts of sexed semen in early trials, 2 sets of heifers in which someturned out to be pregnant up to about 55 days at the time of estrussynchronization, etc. Up to 4 bulls and 3 inseminators were involvedwith each trial; this enabled us to sample populations to ensure thatresults applied to more than one bull or technician; however,insufficient data were produced to evaluate bull-to-bull differences infertility rigorously.

Most sets of heifers were from breeding herds located 140 to 250 km fromour laboratory. There were no significant differences in pregnancy ratesbetween inseminators in any trial, but numbers of breedings perinseminator were low, and differences likely would be detected withlarger numbers of inseminations.

Estrus synchronization methods were not compared within trials, so itwas not possible to compare pregnancy rates among these methods.Pregnancy rates appeared to be satisfactory for all four synchronizationprocedures used.

Since inseminations were done once a day, heifers in estrus eveningswere inseminated approximately 24 h after estrus was detected. Thepregnancy rate for these heifers with sexed sperm pooled over all trialswas 203/414 (49.0%), which was not significantly different (P>0.1) fromthat of heifers in estrus mornings and thus inseminated half a day afterestrus detection 266/586 (45.4%). This tendency for higher fertilitywith later insemination is in agreement with findings from otherresearch that it is preferable to inseminate later than normallyrecommended with lower fertility bulls, when low sperm numbers are used,or when conditions are otherwise suboptimal.

Pregnancy rates by treatments and, when available, fetal or calf sex arepresented in Tables 10 to 20. The objective was to obtain femaleoffspring, except in trial 8; accuracy was 95%, 83%, 90%, 83%, 82%, and94% in Trials 1, 3, 8, 9, 10, and 11, respectively. In the remainder ofthe trials, fetal or birth sexes were not available because of timing ofpregnancy diagnosis, unavailability of persons skilled in sexingfetuses, and/or because calves have not yet been born. This was not amajor concern because the main objective of this research was todetermine fertility of flow-sorted sperm inseminated at low doses.

The accuracy of sexing can be adjusted to virtually any level desiredbetween 50 and 95+% by adjusting the sorting parameters. However, higheraccuracy results in lower numbers of sperm sorted per unit time,particularly for Y-chromosome sperm. 90% accuracy is sufficient forroutine work.

The main findings from each field trial will be summarized in turn. Notethat total sperm numbers are given in table headings; numbers ofprogressively motile sperm usually were 30 to 50% of these values. Fieldtrial 1 (Table 10) confirmed that pregnancy rates with uterine horninsemination using low numbers of unsexed sperm were similar to controlswith normal sperm numbers. The day 64 to 67 pregnancy rate with unfrozensexed sperm (42%) was 12 percentage points below the unsexed liquidcontrol with sperm diluted, stained, and centrifuged identically to thesorted sperm. Accuracy of sexing was 95%; the sex of calves born fromsexed sperm matched the sex diagnosis of fetuses exactly; there was onemistake in sexing fetuses of controls. There were no abortions between 2months of gestation and term, and all 19 calves from the sexed spermtreatment were normal and survived. For the sexed semen treatment, the2-month pregnancy rates for bulls N1, N2, and N3 were 41, 44, and 40%,respectively; 39% (13/33) of heifers in estrus in the morning and 50%(6/12) in estrus in the evening became pregnant.

TABLE 10 Results of field trial 1 - Angus heifers in Wyoming, 1997 No.No. pregnant pregnant Treatment/ No. No. day day No. ♀ site spermheifers 31 to 33 64 to 67 calves Sexed, 3 × 10⁵ 45 20 (44%) 19 (42%) 18(95%)^(a) 5° C./horns Control, 3 × 10⁵ 28 15 (54%) 15 (54%)  5 (53%)^(b)5° C./horns Frozen 40 × 10⁶  29 16 (55%) 15 (52%) 11 (73%)^(a,b)control/body ^(a,b)Sex ratios without common superscripts differ (P <0.02).

Field trial 2 (Table 11) provided the first evidence that results withsexed, frozen sperm are similar to sexed, unfrozen sperm if adjustmentis made for numbers of sperm killed during cryopreservation. There alsowas no difference in pregnancy rates between sexed sperm stored at 5versus 18° C. Pregnancy rates at 2+ months after insemination for sexedsemen from individual bulls ranged from 22 to 42% pregnant (P>0.05).Embryonic loss between 1 and 2 months of gestation was very similar forsexed and control pregnancies. Calving data were available from 39heifers from this trial; each of these heifers (30 sexed pregnancies, 9controls) pregnant at 2 months calved after a normal-length gestation.

TABLE 11 Results of field trial 2--Crossbred beef heifers in Colorado,1998 No. No. No. pregnant No. pregnant Treatment/site sperm heifers day30 to 35^(a) day 59 to 92^(a) Control, 5° C./horns 5 × 10⁵ 58 27 (47%)24 (41%) Sexed, 5° C./horns 5 × 10⁵ 51 17 (33%) 16 (31%) Sexed, 18°C./horns 5 × 10⁵ 46 16 (35%) 12 (26%) Sexed, frozen/horns 1 × 10⁶ 87 29(33%) 28 (32%) ^(a)No significant differences, χ²

Field trial 3 (Table 12) confirmed that sexed, frozen sperm results inreasonable pregnancy rates. The accuracy of sexing sperm was confirmedagain; however, there were 4 errors in sexing fetuses relative to thecalves born; the actual sexes of calves born are presented. Again, therewere no abortions between 2 months of gestation and term. Pregnancyrates averaged over sexed, unfrozen and sexed, frozen sperm for bullsN8, N9, AN5, and AN4 were 24; 31, 50, and 60%, respectively (P<0.1).

TABLE 12 Results of field trial 3 - Angus heifers in Wyoming, 1998 No.No. No. pregnant No. ♀ Treatment/site sperm heifers day 62 to 65 calvesSexed, 18° C./horns 5 × 10⁵ 37 11 (30%)^(a) 10 (91%)^(c) Sexed,frozen/horns 1 × 10⁶ 35 18 (51%)^(a,b) 14 (78%)^(c) Frozen, control/body40 × 10⁶  37 27 (73%)^(b) 16 (59%)^(d) ^(a,b)Means without commonsuperscripts differ P < 0.05). ^(c,d)The percentage of ♀ calves from thesexed treatments (83%) differed from the control group, P > 0.05,1-tail, χ².

Field trials 4, 5, and 6 (Tables 13, 14, 15) were done at the samelocation with 3 different groups of heifers. Unfortunately, it was notpossible to replicate each trial similarly due to vagaries of fieldtrials, such as scheduling personnel, availability of sexed semen fromeach bull, etc. The widely different pregnancy rates between trials 5and 6 illustrate that conditions were different among trials. Some ofthe heifers in trial 6 were available because they failed to getpregnant after a month of natural mating. Under conditions of thesetrials, pregnancy rates were very similar between 1.5 and 3.0×10⁶ sexed,frozen sperm/dose. Furthermore, there was no advantage to uterine-horninsemination. There were no significant differences (P>0.05) inpregnancy rates among bulls except in Trial 5 in which the pregnancyrate of J2, 20/28 (71%), was higher than that of J4, 15/39 (38%)(P<0.05). This difference was not consistent from trial to trial, as J4had numerically but not significantly (P>0.1) higher pregnancy ratesthan J2 in Trials 4 and 6.

TABLE 13 Results of field trial 4-Holstein heifers in Colorado, 1999 No.No. No. pregnant No. pregnant Treatment/site sperm heifers day 30 to 33day 64 to 67* Sexed, frozen/body 1.5 × 10⁶ 55 36 (65%)^(a,b) 36(65%)^(a,b) Sexed, frozen/body   3 × 10⁶ 52 27 (52%)^(a) 26 (50%)^(a)Control, frozen/  20 × 10⁶ 55 45 (82%)^(b) 43 (78%)^(b) body ^(a,b)Meanswithout common superscripts differ (P < 0.01). *Six heifers pregnant atd 30 to 33 were sold before the second pregnancy diagnosis; these wereassumed to have remained pregnant.

TABLE 14 Results of field trial 5 - Holstein heifers in Colorado, 1999No. No. No. pregnant No. pregnant Treatment/site sperm heifers day 33 to35^(a) day 60 to 62^(a) Sexed, frozen/body 1.5 × 10⁶ 23 12 (52%) 12(52%) Sexed, frozen/body 3.0 × 10⁶ 25 15 (60%) 14 (56%) Sexed,frozen/horns 1.5 × 10⁶ 25 15 (60%) 12 (48%) Sexed, frozen/horns 3.0 ×10⁶ 25 17 (68%) 15 (60%) Control, frozen/body  20 × 10⁶ 30 20 (67%) 19(63%) ^(a)No significant differences.

TABLE 15 Results of field trial 6 - Holstein heifers in Colorado, 1999No. No. No. pregnant No. pregnant Treatment/site sperm heifers day 31 to34 day 60 to 63 Sexed, frozen/body 1.5 × 10⁶ 27 11 (41%)^(a) 9 (33%)^(a)Sexed, frozen/body 3.0 × 10⁶ 25 10 (40%)^(a) 9 (36%)^(a) Sexed,frozen/horns 1.5 × 10⁶ 24  8 (33%)^(a) 7 (29%)^(a) Sexed, frozen/horns3.0 × 10⁶ 24 10 (42%)^(a) 8 (33%)^(a) Control, frozen/body  20 × 10⁶ 2418 (75%)^(b) 17 (71%)^(b)  ^(a,b)Means without common superscriptsdiffer (P < 0.05).

For trial 7 (Table 16), only one inseminator was available due torescheduling. This is the only trial that showed a convincing advantageof uterine-horn over uterine-body insemination. For this inseminatorunder the conditions of the trial, 55% more heifers (22 percentagepoints) became pregnant with sexed, frozen semen inseminated into theuterine horns than into the uterine body. The true difference could besmaller because there are wide confidence intervals on these means. Inall the other trials (5, 6, 9, and 11) in which body- andhorn-insemination were compared, pregnancy rates were very similar forboth methods for this technician as well as for other technicians.

Semen from one of the bulls used in Trial 7 was shipped without dilutionfrom Montana by air in an insulated box at −20° C. before sorting;shipping time was 6 h. Pregnancy rates for the sexed sperm from the twobulls were virtually identical, 49% for the unshipped and 52% for theshipped semen. Semen was not diluted with extender and not cooled forshipping because staining properties of sperm with Hoechst 33342 arealtered by dilution with extenders. Furthermore, in other studies (seeExample 4), storing semen neat at ambient temperature between collectionand flow-sorting was found to be superior to diluting it.

TABLE 16 Results of field trial 7 - Crossbred beef heifers in Colorado,1999 No. No. No. pregnant Treatment/site sperm heifers day 33 to 37Sexed, frozen/body 1.5 × 10⁶ 86 34 (40%)^(a) Sexed, frozen/horns 1.5 ×10⁶ 86 53 (62%)^(b) Control, frozen/body  20 × 10⁶ 35 18 (51%)^(a,b)^(a,b) Means without common superscripts differ (P < 0.01).

Field trial 8 (Table 17) concerned feedlot heifers not implanted withgrowth promotants; at the time pregnancy was diagnosed they wereaborted, so calving data was not available. This experiment illustratesthat efficacious sexing also can be done in the male direction.Pregnancy rates for the 2 bulls were 50 and 61%.

TABLE 17 Results of field trial 8 - Angus heifers in Nebraska, 1999 No.No. No. pregnant^(a) No. ♂ Treatment/site sperm heifers day 74 to 76fetuses Sexed, frozen 72 mW 1 × 10⁶ 18  7 (39%)  6 (86%) laser/bodySexed, frozen, 135 mW 1 × 10⁶ 18 13 (78%) 12 (92%) laser/body ^(a)Nosignificant differences.

Field trial 9 (Table 18) was the only trial to show a convincingadvantage of 3.0 versus 1.5×10⁶ sexed, frozen sperm/insemination dose.This advantage was true for both inseminators. Pregnancy rates for sexedsperm from the 2 bulls were 62 and 75%.

TABLE 18 Results of field trial 9 - Red Angus heifers in Nebraska, 1999No. No. No. pregnant No. ♀ Treatment/site sperm heifers day 60 to 63^(a)fetuses Sexed, frozen/body 1.5 × 10⁶ 15  8 (53%) 7 (88%) Sexed,frozen/body 3.0 × 10⁶ 14 12 (86%) 9 (75%) Sexed, frozen/horns 1.5 × 10⁶16  9 (56%) 7 (78%) Sexed, frozen/horns 3.0 × 10⁶ 16 12 (75%) 11 (92%) Control, frozen/body  20 × 10⁶ 30 21 (70%) 13 (62%)  ^(a)3.0 × 10⁶ sexedsperm had a higher pregnancy rate (80%) than 1.5 × 10⁶ sexed sperm(55%), P < 0.05, 1-tail χ².

Pregnancy rates in field trial 10 (Table 19) with sexed, frozen semen,were similar to controls; the accuracy of sexing sperm on this trial wasonly 82%, which, however, is not significantly different from thetargeted 90% accuracy. Pregnancy rates for sexed semen were 54, 66, and50% for bulls AN4, AN7, and AN8, respectively (P>0.1). Eighteen of theheifers inseminated in this trial were the calves resulting from sexedsperm in field trial 1.

TABLE 19 Results of field trial 10 - Angus heifers in Wyoming, 1999 No.No. No. pregnant No. ♀ Treatment/site sperm heifers day 61 to 63^(a)fetuses Sexed, frozen/body   1 × 10⁶ 44 26 (59%) 23 (85%) Sexed,frozen/body 3.0 × 10⁶ 43 23 (53%) 17 (74%) Control, frozen/body  20 ×10⁶ 35 20 (57%) 12 (57%) ^(a)No significant differences.

TABLE 20 Results of field trial 11 - Holstein heifers in Colorado, 1999No. No. No. pregnant No. pregnant Treatment/site sperm heifers day 28 to30^(a) day 56 to 58^(a,b) Sexed, frozen/body 1 × 10⁶ 12 8 (67%) 7 (58%)Sexed, frozen/body 3 × 10⁶ 12 6 (50%) 4 (33%) Sexed, frozen/horns 1 ×10⁶ 7 4 (57%) 4 (57%) Sexed, frozen/horns 3 × 10⁶ 7 4 (57%) 4 (57%)Control, frozen/ 20 × 10⁶  9 4 (44%) 3 (33%) body ^(a)No significantdifferences, χ². ^(b)16 of 17 (94%) fetuses from the sexed sementreatments were female; 2 were too deep in the body cavity to sex withultrasound.

Data from trials were combined in which treatments were identical except1×10 and 1.5×10⁶ sperm doses were pooled (Table 21).

TABLE 21 Meta-summary from combining trials with sexed, frozen semen andfrozen controls. Trials combined Sperm no./site No. heifers No. pregnant5, 6, 9, 11 1.0-1.5 × 10⁶/body 77 36 (47%) 3.0 × 10⁶/body 76 38 (50%)1.0-1.5 × 10⁶/horns 72 32 (44%) 3.0 × 10⁶/horns 72 39 (54%)  20 ×10⁶/body, control 93 61 (66%) 4, 5, 6, 9, 10, 11 1.0-1.5 × 10⁶/body 17698 (56%) 3.0 × 10⁶/body 171 88 (51%)  20 × 10⁶/body, control 183 124(68%)  5, 6, 7, 9, 11 1.5 × 10⁶/body 163 70 (43%) 1.5 × 10⁶/horn 158 85(54%)  20 × 10⁶/body, control 128 79 (62%)

Pregnancy rates with sexed sperm were generally 70-90% of unsexedcontrols within experiments with 7 to 20 times more sperm. Thisdifference was less in the more recent trials, possibly reflectingimproved sexing and sperm-processing procedures.

In some trials, heifers were examined for pregnancy by ultrasound atboth 1 and 2 months after insemination. Pregnancy losses in thisinterval were similar (P>0.1) for sexed (23/261; 8.8%) versus control(9/145; 6.2%) sperm treatments, which is one measure that genetic damagedue to sexing is minimal. Calving information was obtained from only afew of the pregnant heifers because most cattle from the earlier trialswere sold, and those from later trials have not calved yet. Thepopulation of calves produced to date from sexed semen appears to be nodifferent from the population of controls.

CONCLUSION

Sex ratios in cattle can be distorted to about 90% of either sex bysorting sperm on the basis of DNA content with a flow cytometer/cellsorter followed by cryopreservation and relatively routine artificialinsemination. Calves resulting from sexed sperm appear to be normal. Formost bulls in these studies, pregnancy rates with 1.0 to 1.5×10⁶ sexed,frozen sperm were 70 to 90% of unsexed controls with 20 or 40×10⁶ frozensperm inseminated conventionally. These results apply to well-managedheifers bred by well-trained inseminators using properly processedsemen. There may be a small advantage to inseminating sexed spermbilaterally into the uterine horns compared to standard uterine bodyinsemination.

The present invention has of necessity been discussed herein byreference to certain specific methods and materials. It is to beunderstood that the discussion of these specific methods and materialsin no way constitutes any limitation on the scope of the, presentinvention, which extends to any and all alternative materials andmethods suitable for accomplishing the ends of the present invention.

All patents and publications described are herein incorporated byreference in their entirety.

1-55. (canceled)
 56. A method of cryopreserving selected sperm cellscomprising: a) obtaining selected sperm cells; b) cooling the selectedsperm cells; c) extending the selected sperm cells with an initialextender; d) isolating the selected sperm cells; e) extending theisolated sperm cells with a final extender to produce a spermsuspension; and f) freezing said sperm suspension.
 57. The method ofclaim 56, wherein the step of obtaining selected sperm cells furthercomprises: a) staining a mixed population of sperm cells with aflourochrome dye which binds to sperm DNA; b) differentiating Xchromosome bearing sperm from Y chromosome bearing sperm through flowcytometry based on an assessment of the amount of DNA in each cell; andc) separating sperm to produce a selected sperm cells which are enrichedwith respect to the presence of either X chromosome bearing sperm or Ychromosome bearing sperm.
 58. The method of claim 56, wherein the frozensperm suspension, when thawed, comprises a sufficient number of spermcells to fertilize an egg.
 59. The method of claim 56, wherein the finalextender comprises one selected from the group consisting of: anegg-yolk-Tris extender containing glycerol; egg-yolk-TES-Tris extendercontaining glycerol; an egg-yolk-sodium citrate extender containingglycerol; and a milk extender containing glycerol.
 60. The method ofclaim 56, wherein the final extender comprises a cold shock treatment,an energy source, an antibiotic, a component which maintains osmolalityand buffers pH, and a cryoprotectant.
 61. The method of claim 60,wherein the energy source is selected from the group consisting of: asaccharide, a glucose, a fructose, and any combination thereof.
 62. Themethod of claim 60, wherein the cold shock treatment is selected fromthe group consisting of: egg yolk, an egg yolk extract, milk, a milkextract, casein, albumin, lecithin, and any combination thereof.
 63. Themethod of claim 60, wherein the cryoprotectant is selected from thegroup consisting of glycerol, dimethyl sulfoxide, ethylene glycol,propylene glycol, and any combination thereof.
 64. The method of claim63, wherein the cryoprotectant comprises about 5% to about 7% glycerol.65. The method of claim 60, wherein the component which maintainsosmolality and buffers pH is selected from the group consisting of: abuffer comprising a salt, a buffer containing a carbohydrate, and anycombination thereof.
 66. The method of claim 60, wherein the componentwhich maintains osmolality and buffers pH is selected from the groupconsisting of sodium citrate, Tris[hydroxymethyl]aminomethane, TESN-Tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid, monosodiumglutamate, milk, HEPES buffered medium, and any combination thereof. 67.The method of claim 56, wherein, prior to freezing, the sperm suspensionis divided into individual insemination samples.
 68. The method of claim67, wherein the individual insemination samples comprises between aboutone million and about three million selected sperm cells.
 69. The methodof claim 56, wherein the sperm cells are selected from the groupconsisting of: bovine sperm cells, equine sperm cells, and porcine spermcells.
 70. The method of claim 56, wherein the step of cooling theselected sperm cells comprises the step of reducing the temperature ofsaid sperm cells to about 5° Celsius.
 71. The method of claim 70,wherein the step of reducing the temperature of said sperm cells toabout 5° Celsius comprises reducing the temperature for a period betweenabout 60 minutes and about 240 minutes.
 72. The method of claim 56,wherein the initial extender comprises a cold shock treatment, an energysource, an antibiotic, and a component which maintains osmolality andbuffers pH.
 73. The method of claim 56, wherein the selected sperm cellsare sorted into an initial extender that lacks glycerol and wherein thestep of extending the selected sperm cells further comprises extendingthe selected sperm cells in an initial extender having a cryoprotectant.74. The method of claim 73, wherein the step of extending the selectedsperm cells with an initial extender having a cryoprotectant furthercomprises extending applying to equal volumes of the initial extenderhaving a cryoprotectant about 15 minutes apart.
 75. A frozen separatedcell sample in ccordance with the method of claim 56.